1

Oxidative Stress in Hostile Environments:

A Translational Omics-Based Approach to

Personalized Assessment &

Countermeasures Development

Thomas J. Goodwin, Ph.D.NASA Johnson Space Center

Lead, Translational Research and Medicine

Oxidative Stress and Damage

&

Michael A. Schmidt, Ph.D.MetaboLogics, LLC

Advanced Pattern Analysis and Countermeasures Group

https://ntrs.nasa.gov/search.jsp?R=20150016014 2019-07-06T23:13:53+00:00Z

Oxidative Stress and Damage

A Common Denominator In Space Flight Health?

KOHEN AND NYSKA

J. TOXICOLOGIC PATHOLOGY, Vol. 30, 2002

REDOX Balance

Point

Causal Factors/ Risks in the Production of OSaD

KOHEN AND NYSKA

J. TOXICOLOGIC PATHOLOGY, Vol. 30, 2002

Environmental Physiological

Weightlessness

High Proton Radiation

Illustrates specific free radical (ROS) species (red circles) and ions that contribute to loss of

REDOX homeostasis and cellular damage, inflammation, and DNA damage. (Adapted

from Kohen R, Nyska A. Toxicol Pathol, 2002) 4

Sources of ROS

Oxidative Stress and Damage Potentially

Related to Space Flight Phenomena

We hypothesize that OSaD may be a contributing factor in the

following areas of space flight related dysregulations:

1) Cardiovascular effects (Atherosclerosis)

2) Intracranial hypertension (VIIP)(Cephalic Fluid Shift)

3) Immune dysregulation and suppression

4) Bone loss and potential fragility

5) Hypoxia and Hyperoxia (DCS)

6) Altered one carbon metabolism linked to single

nucleotide polymorphisms (SNPs)

7) Other relevant SNPs (e.g. HFE)

Causal Factors that Occur in 1G that also May Relate to OSaD in Space Flight

Courtesy National Institute of Standards and Technology (NIST)Courtesy of NASA/JSC Education and Outreach

One Primary Concern in Space: Protecting Against DNA Damage

7

(Image Credit: NASA (assumed), via ITECS Insider)

Space Radiation

In Addition to OSaD What if…

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DNA is Unstable BEFORE Entering Space

What New Countermeasures are Possible?

Two Attributes of DNA Integrity

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DNA Stability

DNA RepairFigures adapted from Puranik, M.

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DNA Stability

Michael A Schmidt, PhD & Thomas J Goodwin, Ph.D. Copyright ©2013

DNA Stability: Gene & Micronutrient Influence

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Genetics (MTHFR, MTR, etc.)

Micronutrients (folate, B12, etc.)

DNA Stability: Uracil Misincorporated into DNA Strand

12

From: Schmidt, MA, Goodwin, TJ. (2013)

Personalized Medicine in Human Space Flight.

Metabolomics.

Genetics (MTHFR, MTR, etc.)

Micronutrients (folate, B12, etc.)

DNA Stability: If poor methyl status, uracil accumulates

13From: Schmidt, MA, Goodwin, TJ. (2013) Personalized Medicine in Human Space Flight. Metabolomics.

Radiation strikes unstable

DNA-Mutational Event 2

Uracil > unstable DNA-

Mutational Event 1

DNA Stability: High Uracil, Folate Corrects

14From: Schmidt, MA, Goodwin, TJ. (2013) Personalized Medicine in Human Space Flight. Metabolomics.

• Blount et al.: Uracil levels were found to be 70 times higher in

subjects whose serum folate < 4 ng/ml (as high as 4M uracil

residues/cell).

• After three days supplementation with 5 mg folic acid, uracil

levels were rapidly reduced. (Roughly 12x RDI of 400mcg/d)• RDI=Recommended Daily Intake for Healthy Individuals

• Are Healthy individuals still health in Space?

Blount, B. C., et al. (1997). Proc Natl Acad Sci U S A, 94, 3290-3295.

DNA Stability: If poor methyl status, damage similar to and possibly compounded by elevated radiation

15

From: Schmidt, MA, Goodwin, TJ. (2013) Personalized Medicine in Human Space Flight. Metabolomics. In Press

• Chromosomal damage caused by reducing folate concentration

from 120 to 12 nmol/L

• Equivalent to that induced by an acute exposure to 0.2 Gy of

low linear-energy-transfer ionizing radiation (e.g., X-rays)

• A dose of radiation that is 10 times greater than the annual

allowed safety limit of exposure for the general population

Fenech, M. (2010) Am J Clin Nutr, 91, 1438S-1454S.

Fenech, M. (2012) Mutation Research, 733:21-33.

Gene & Micronutrient Variants

Reduce DNA Stability

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Courtemanche, C., Huang, A. C., Elson-Schwab, I., et al. (2004). Folate deficiency and ionizing radiation cause DNA breaks

in primary human lymphocytes: a comparison. FASEB J, 18, 209-211.

Fenech, M. (2012) Folate (vitamin B9) and vitamin B12 and their function in the maintenance of nuclear and mitochondrial

genome integrity. Mutation Research, 733:21-33.

Schmidt, MA, Goodwin, TJ. (2013) Personalized Medicine in Human Space Flight. J. Metabolomics.

Are One Carbon Deficits

Common in Astronauts?

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Factor OC+ Finding

Metabolite Homocysteine Methylmalonic AcidCystathionine

ElevatedElevatedElevated

Essential Input (Nutrient)

Folic AcidB12B6

Low in 4/5 OC+

LowLow (not signif)

Genotype (MTHFR, MTRR, etc)

Unknown Unknown

Smith, SM, et al. Vision Changes after Spaceflight Are Related to Alterations in Folate– and

Vitamin B-12–Dependent One-Carbon Metabolism. J. Nutr. doi: 10.3945/jn.111.154245.

ISS Study: Those with (OC+; n=5) and without (OC-;n=15) ophthalmic changes

How Common Are One Carbon Variants? Widespread in Population

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CC (n) CT (n) TT (n)

MTHFR C677T

1298AA 1492 2217 783

1298AC 2469 1848 16

1298CC 1246 43 0

86% of 10,601 people possessed at least one mutant MTHFR allele (MTHFR C677T

and A1298C). Fully 17.4% were heterozygous for both MTHFR C677T and A1298C.

Fredriksen, A, Meyer, K, Ueland, PM, et al. Large-scale population-based metabolic phenotyping of thirteen genetic

polymorphisms related to one-carbon metabolism. Human Mutation 2007;28(9):856-65.

Folate, B12: Keeping DNA Stable

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Prevent Anemia Minimize DNA Damage

Plasma Folate (ng/ml) 2.2 21 (7.3-53.0)

RBC Folate (ng/ml) 132 464 (313-600)

Prevent Anemia Minimize DNA Damage

Plasma B12 (pmol/L) 150 400

Beck, S, Olek, A. The Epigenome, 2003

Fenech et al. Carcinogenesis, 1998;19:1163-71; Fenech 2012

Schmidt, MA, Goodwin, TJ (2013) Personalized Medicine in Human Space Flight. Metabolomics In Press

Michael A Schmidt, PhD & Thomas J Goodwin, Ph.D. Copyright ©2013

20

DNA Repair and Energy

Production

Michael A Schmidt, PhD & Thomas J Goodwin, Ph.D. Copyright ©2013

DNA Repair: Mg Dependence

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Figures adapted from Puranik, M.

• Mg is important for maintaining the integrity of

DNA

-Anastassopoulou, J. et al. (2002) Magnesium-DNA interaction and the possible relation of magnesium to carcinogenesis. Irradiation

and free radicals. Crit. Rev. Oncol. Hematol., 42, 79–91.

-Hartwig, A. (2001) Role of magnesium in genomic stability. Mutat. Res., 475, 113–121.

• Mg cations (Mg++) bind to DNA and reduce the

negative charge density, thereby stabilizing the

structure of DNA

• Mg is cofactor for the enzymes:

- nucleotide excision repair

- base excision repair

- mismatch repair (MMR)

• MMR activity lowers the mutation

frequency in the genome by 2–3 orders of

magnitude

• DNA repair REQUIRES ATP

Magnesium-ATP Complex

Magnesium-ATP

complex is the sole

biologically active

form of ATP

All ATP

reactions

involve Mg

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Magnesium-ATP Synthase

ATP Synthase also

requires

magnesium

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JBC May 12,

2006 vol. 281

no. 19 13777-

13783

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Lebbink JH, et al. Magnesium Coordination Controls the Molecular Switch Function of DNA

Mismatch Repair Protein MutS* J. Biol. Chem. 2010:285(17);13131-41.

Magnesium Controls DNA Mismatch Repair Switch:

Needs ATP

After 6 months in space:

Loss of Mg reservoirs

35% loss in some leg muscles

~1-2% average loss per month in bone

Decreased plasma volume ~15-20%

Loss precipitated by reduced gravity, Cephalic fluid shifts, and stress related reactions to confinement

• Smith, S. M., et al. (2005). J Bone Miner Res, 20, 208-218.

• Smith, S. M., et al (2005). J Nutr, 135, 437-443.

• Fitts RH, et al. J Physiol 2010;588(Pt 18):3567–3592

Urinary Mg levels:

44% lower after landing than before launch (P< 0.001)

55% of ISS crew members had Mg concentrations lower than the low end of the clinical range (3.0 mmol/d)

Question: Are we flying astronauts with diminished DNA repair capacity Unknowingly?

• Smith, S. M., et al. (2005). J Bone Miner Res, 20, 208-218.

• Smith, S. M., et al (2005). J Nutr, 135, 437-443.

• Fitts RH, et al. J Physiol 2010;588(Pt 18):3567–3592

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Convergent Influences in

Space Medicine

Unstable DNA + Altered DNA Repair

+ Radiation

Convergent InfluencesOpportunity for Countermeasures

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Astronaut Safety &

Performance

Unstable DNA

Many Variants

RadiationHostile

Environment

Altered DNA Repair

Many Variants

(Image Credit: NASA (assumed),

via ITECS Insider)

Convergent InfluencesOpportunity for Countermeasures

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Astronaut Safety &

Performance

Radiation

SNPs: One Carbon

• MTHFR (C677T, A1298C)

MTR (A2756G)

MTRR (A66G)

BHMT (G742A)

CBS (C699T)

TCN2 (C776G)

Mg: DNA Repair• DNA glycosylases• AP endonucleases• End processing enzymes• DNA polymerases• Flap endonuclease• DNA ligase• Mismatch Repair• Base excision repair• ATP synthase (All)• ATP stabilization (All)

Unstable DNA

Many Variants

Altered DNA Repair

Many Variants

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Using Omics to Drive Research & Personalize Care

From: Schmidt, MA, Goodwin, TJ. (2013) Personalized Medicine in Human Space Flight: Using Omics Based Assessment

to Develop Individualized Countermeasures that Enhance Astronaut Safety and Performance. J. Metabolomics.

Orbital